Although many analogs of vitamin D metabolites have been synthesized and evaluated for their biological activity, very little is known about the mechanisms responsible for the observed structure-activity relations for these compounds. We have begun a research program that will critically analyze the important structural features of the vitamin D3 molecule that are required for its metabolism and biological activity. To study the metabolism of 1alpha,25-(OH)2-D3 and to investigate what effect a change in the stereochemical position of the C1-hydroxyl function has on the biological activity of 1,25-(OH)2-D3, we chemically synthesized (1alpha-3H)1beta,25-(OH)2-D3, (1beta-3H)1alpha,25-(OH)2-D3 and 1beta,25-(OH)2-D3. This was accomplished by oxidizing 1alpha,25-(OH)2-D3 with MnO2 to I-oxo-25-hydroxyprevitamin D3. This oxidation product was subsequently reduced with NaBH4 or 3H-NaBH4 to yield a mixture of 1beta,25-(OH)2-D3 and 1alpha, 25-(OH)2-D3 (and their tritium labeled counterparts) in a ratio of 93:7. During the next year we plan to carefully evaluate the biological activity and metabolism of 1beta,25-(OH)2-D3 as a model system to continue our probing of the structure-function relationships of the vitamin D3 molecule.